In an infected cell, Hepatitis B virus (HBV) cores assemble in the cytoplasm. The cores are comprised of a reverse transcriptase-pregenomic RNA complex and associated chaperonins contained in an icosahedral capsid of core protein (Cp). After the RNA is reverse transcribed, the now DNA-filled cores are directed to either the nucleus to maintain HBV infection or to the ER to be secreted. All of these steps require interaction with host proteins. To package the correct RNA, the Cp must be phosphorylated. DNA-filled cores that are transported to the nucleus are phosphorylated while secreted virions are dephosphorylated, indicating roles for kinases and phosphatases. For either fate, DNA-filled cores must be actively transported. We hypothesize that the HBV capsid is a highly dynamic structure that transiently displays occluded segments (phosphorylation sites, nuclear localization signals, late domains) in response to internal and external stimuli. In this R21 application we will investigate HBV Cp interaction with two host proteins believed to be critical for intracellular activity. The Ser-Arg directed protein kinase SRPK has been shown to phosphorylate the Cp RNA-binding domain, which is on the interior of capsids. In aim 1, we will investigate the interaction of SRPK with empty and nucleic acid-filled capsid. In aim 2, we will examine interaction of capsids with NEDD4, an E3 ubiquitin ligase that is important for regulating trafficking of HBV particles. NEDD4 interacts with most proteins by binding to PPxY sequences;in HBV capsids this sequence is buried at a protein-protein interface but is transiently exposed by capsid breathing. For both aims, we will examine binding using enzymatic activity, column assays, and surface plasmon resonance. Binding of both SRPK and NEDD4 to capsids is likely to require capsid breathing and distortion of capsid quaternary structure. We will examine how capsid stability and contents relate to binding activity, making a connection between virus maturation and trafficking. These experiments will lead to examination of other host partners and the physical and structural basis of regulating their activities with HBV. By identifying host partners and their mechanism of binding, we identify new targets for antiviral therapeutics within the host cell and on the core of the virus. PUBLIC HEALTH RELEVANCE: Chronic Hepatitis B virus (HBV) infection is a major risk factor for cirrhosis of the liver and hepatocellular carcinoma, contributing to 1 million deaths per year. Like all viruses, HBV hijacks components of host cells to regulate virus assembly, maturation, and secretion. We will investigate how HBV interacts with two host proteins that are critical to HBV replication and may represent new targets for antiviral therapeutics.